Macrophages Regulate the Angiogenic Switch in a Mouse Model of Breast Cancer Elaine Y. Lin, 1 Jiu-Feng Li, 1 Leoid Gnatovskiy, 1 Yan Deng, 2 Liyin Zhu, 1 Dustin A. Grzesik, 2 Hong Qian, 3 Xiao-nan Xue, 3 and Jeffrey W. Pollard 1 1 Department of Developmental and Molecular Biology, Center of Reproductive Biology and Women’s Health, 2 Analytical Imaging Facility, and 3 Department of Epidemiology and Population Health, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York Abstract The development of a tumor vasculature or access to the host vasculature is a crucial step for the survival and metastasis of malignant tumors. Although therapeutic strategies attempting to inhibit this step during tumor development are being developed, the biological regulation of this process is still largely unknown. Using a transgenic mouse susceptible to mammary cancer, PyMT mice, we have characterized the development of the vasculature in mammary tumors during their progression to malignancy. We show that the onset of the angiogenic switch, identified as the formation of a high- density vessel network, is closely associated with the transition to malignancy. More importantly, both the angiogenic switch and the progression to malignancy are regulated by infiltrated macrophages in the primary mammary tumors. Inhibition of the macrophage infiltration into the tumor delayed the angiogenic switch and malignant transition whereas genetic restoration of the macrophage population specifically in these tumors rescued the vessel phenotype. Furthermore, premature induction of macrophage infiltration into premalignant lesions promoted an early onset of the angiogenic switch independent of tumor progression. Taken together, this study shows that tumor-associated macrophages play a key role in promoting tumor angiogenesis, an essential step in the tumor progression to malignancy. (Cancer Res 2006; 66(23): 11238-46) Introduction Tumor progression is characterized by an initial ‘‘avascular phase’’ when the tumors are small and usually dormant (1) with diffusion being the major way to support their metabolic needs (2). In the subsequent ‘‘vascular phase,’’ the development of a unique tumor vasculature is required for the increased metabolic demand of tumors that have grown beyond a certain size. The induction of this vasculature, termed the ‘‘angiogenic switch’’ (1, 3, 4), can occur at various stages of tumor progression, depending on the tumor type and the environment (1). However, it is clear that malignant tumors require its development as it has been shown that the initiation of revascularization in dormant lesions allows them to progress (5, 6). The stroma of solid tumors are replete with many leukocytic cells of which macrophages represent a major component (7). Recent clinical and experimental studies have indicated that these tumor-associated macrophages promote the progression to malignancy (7, 8). In human breast cancers, macrophages cluster in ‘‘hotspots’’ in avascular areas in human breast cancer samples (9), which correlates with a high level of angiogenesis and with decreased relapse-free and overall survival of the patients (10). Macrophages play a crucial role in regulating angiogenesis in wound healing (11). They produce many proangiogenic factors including vascular endothelial growth factor (VEGF), tumor necrosis factor a, granulocyte macrophage colony-stimulating factor, interleukin (IL)-1, IL-6 (11), and other factors including matrix metalloproteinases (MMP) and nitric oxide (12, 13) that also have the potential to regulate angiogenesis (7, 11). Parallels have been drawn between the microenvironment of wound-induced inflammation and that of tumors, as proposed in the hypothesis that tumors are ‘‘wounds that never heal’’ (14). However, whether tumor-associated macrophages are able to promote angiogenesis is still not clear. We have reported in the mouse model of breast cancer caused by the mammary epithelial cell restricted expression of the Polyoma middle T oncoprotein (PyMT mice) that the infiltration of macrophages in primary mammary tumors was positively asso- ciated with tumor progression to malignancy (8). Depletion of macrophages in this model severely delayed tumor progression and dramatically reduced metastasis whereas an increase in macro- phage infiltration by transgenic means remarkably accelerated these processes (8). To identify the mechanism(s) that macro- phages use to promote tumor progression, we have tested the hypothesis that tumor-associated macrophages stimulate the deve- lopment of tumor vasculature. Our results indicate that tumor- associated macrophages were actively involved in promoting the angiogenic switch during the malignant transition as well as in the maintenance and/or remodeling of an established vessel network in malignant tumors. Materials and Methods Mice. All procedures involving mice were conducted in accordance with NIH regulations about the use and care of experimental animals. The study of mice was approved by the Albert Einstein College of Medicine animal use committee. The PyMT transgenic mice and mice carrying the CSF-1R-GFP transgene were kindly provided by Drs. W.J. Muller (McGill University, Montreal, Quebec, Canada) and David Hume (University of Brisbane, Brisbane, Australia), respectively. The origin, care, and identification of CSF-1 null mutant (Csf1 op /Csf1 op ) mice have previously been described (8). Because +/Csf1 op mice have normal serum concentration of CSF-1, normal tissue population of macrophages, and are in all aspects tested equivalent to wild-type (+/+) mice (15), these +/Csf1 op are used as controls. The preparation of CSF-1-expressing transgenic mice has previously been described (8). The mice used were in a mixed genetic background of C3H/ B6/FVB. The genotype of the CSF-1R-GFP mice was determined by directly Requests for reprints: Jeffrey W. Pollard, Albert Einstein College of Medicine, 607 Chanin Building, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-2090; Fax: 718-430-8663; E-mail: pollard@aecom.yu.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1278 Cancer Res 2006; 66: (23). December 1, 2006 11238 www.aacrjournals.org Research Article Research. on May 3, 2016. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst November 17, 2006; DOI: 10.1158/0008-5472.CAN-06-1278